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Part:BBa_K302012:Experience

Designed by: Rachel May Boyd, Phil Hall   Group: iGEM10_Newcastle   (2010-08-12)
Revision as of 11:52, 27 October 2010 by RachelBoyd (Talk | contribs) (→‎Characterisation by Team Newcastle 2010)

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Applications of BBa_K302012

Characterisation by Team Newcastle 2010

We integrated our part into the Bacillus subtilis 168 chromosome at amyE (using the integration vector pGFP-rrnB) and selected for integration by testing for the ability to hydrolyse starch. Homologous recombination at amyE destroys endogenous expression of amylase. Colonies that are not able to break down starch on agar plate do not have a white halo when exposed to iodine.

The part was co-transcribed with gfp fluorescent marker by transcriptional fusion after the yneA coding sequence.

We characterised the part first without, and then with, LacI repression (using the integration vector pMutin4 to integrate lacI into the Bacillus subtilis 168 chromosome).


Figure1:
Distribution of cell lengths is not normal, so the mean is misleading; we are reporting the median instead.
Teamnewcastle yneA168.png
Figure1: shows statistics for populations of cells
  • overexpression of the yneA construct (ΔamyE:pSpac(hy)-oid::yneA(cells with YneA construct but no inhibitory regulation) ) leads to a longer cell length compared with our control Bacillus subtilis 168.
  • pMT4_0.0: YneA construct in pMutin4 vector with inhibition and no IPTG (ΔamyE:Pspac(hy)-oid::yneA::pMutin4)
  • pMT4_1.0: YneA construct in pMutin4 vector with inhibition and 1.0 μM IPTG (ΔamyE:Pspac(hy)-oid::yneA::pMutin4)
with inhibition cell lengths are comparable to Bacillus subtilis 168 at 0μM IPTG and longer with IPTG induction.
See below: Bacillus subtilis 168 cells (left),Bacillus subtilis expressing yneA(centre) and Bacillus subtilis overexpressing yneA(right)
Teamnewcastle yneA168BS.jpgTeamnewcastle yneA1.jpgTeamnewcastle yneA.jpg
The images we have taken this data from had very different numbers of cells, so the cells counts are misleading therefore we are reporting the proportions of cells at a given length.
Graph2:
Newcastle no induction.jpg
Graph 2 shows the percentage of cells at different lengths(μm)uninduced
See below: Bacillus subtilis 168 cells (left) and non-induced cells(right)
Teamnewcastle yneA168BS.jpgTeamnewcastle noindBS.jpg
Graph3:
Newcastle 0.2 induction.jpg
Graph 3 shows the percentage of cells at different lengths(μm)induced at 0.2mM IPTG
See below: Bacillus subtilis 168 cells (left) and cells induced at 0.2mM IPTG(right)
Teamnewcastle yneA168BS.jpgTeamnewcastle 0.2indBS.jpg
Graph4:
Newcastle 1IPTG.jpg
Graph 4 shows the percentage of cells at different lengths(μm)induced at 1mM IPTG
See below: Bacillus subtilis 168 cells (left) and cells induced at 1mM IPTG(right)
Teamnewcastle yneA168BS.jpgTeamnewcastle 1indBS2.jpg

Graphs 2,3 and 4 show a greater proportion of cells at a higher concentration of IPTG(1mM IPTG), compared with Bacillus subtilis 168 our control population.

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